Recent evidence shows that the human genome contains approximately 35,000 different genes. Due to post-translational modifications, the human “proteome,” or the number of proteins produced by these genes, probably numbers in the hundreds of thousands. Much study and effort will be needed to unravel the complete complement and function of the human proteome.
A basic tool for analyzing proteins from the human proteome (and other sources) is electrophoresis. Electrophoresis is a method by which molecules are moved through a porous support or substrate by the application of an electric current. Using this method, a mixture of charged molecules can be separated on the basis of their physical characteristics (e.g., molecular weight or “Mr”) and/or their chemical nature (e.g., charge or isoelectric point) by movement of charged species through an electrolytically conductive medium.
One dimensional (“1-D”) electrophoresis is a standard technique in which molecules are forced to migrate along one axis in a separation substrate. 1-D electrophoretic analysis of proteins is typically performed in a gel matrix (such as polyacrylamide) under denaturing conditions; i.e., using ionic or non-ionic detergents. The detergent used to denature the proteins induces a random configuration and can impart a relatively constant charge/mass ratio to the protein molecules. Under these conditions, the relative mobility of the denatured protein decreases almost linearly with an increase in log(Mr). If protein binding or biological activity is to be preserved, the electrophoresis can be performed under “non-denaturing” conditions, which allow the proteins to retain their native form. Under non-denaturing conditions, relative mobility of proteins is a function of both Mr and charge. However, the resolution of proteins electrophoresed through gels under non-denaturing conditions is usually poor.
Two-dimensional polyacrylamide gel electrophoresis (“2-D PAGE”), first developed by O'Farrell (J. Biol. Chem. 250:4007-4021, 1975), is another widely used method for separating and analyzing proteins. In this method, proteins are separated in the first dimension according to their isoelectric points in the presence of pH gradient generated by using ampholytes or similar materials. This is followed by separating the proteins according to their molecular weights in a second dimension. The proteins are typically electrophoresed in the first dimension under denaturing conditions which do not impose a uniform charge/mass ratio; for example, in the presence of 9M urea. Electrophoresis in the second dimension is typically performed in the presence of an ionic detergent such as SDS, and is analogous to the denaturing 1-D electrophoresis discussed above. Protein-protein interactions or biological activities of the separated proteins are not preserved in conventional 2-D PAGE techniques.
A major impediment in the progress of “proteomics,” or the analysis of the functions of proteins in a cell, is the complexity and the length of time required for the separation of protein molecules. The conventional 2-D PAGE techniques mentioned above involve multiple steps and generally take one to two days to complete. For example, a typical 2-D PAGE protocol includes: 1) preparation of a gel matrix with a specific pH gradient for performing the first dimensional isoelectric focusing (IEF) step, and the “running” of the IEF gel in the first dimension; 2) equilibration of the IEF gel in the buffer used for the second dimension run, and 3) the transfer of proteins from the IEF gel onto the second dimension slab gel and subsequent running of the second dimension electrophoresis. Although preformed IEF gel strips with a specific pH gradient are commercially available, such strips are typically provided dry and require a rehydration step of 10-12 hours prior to use.
Efficient separation of molecules by 1-D or 2-D electrophoresis requires that all molecules of the same substance have equal velocity during the separation process. To achieve this, the electric field, and therefore the conductivity, must be uniform throughout the volume of the separation medium. The conductive characteristics of the molecules being separated, however, cause the conductivity of the medium to become nonuniform. In practice, a uniform electric field is approximated by using an electrophoretic separation medium and buffer with a high conductivity relative to the conductivity contribution of the molecules being separated. Highly conductive electrophoretic separation media and buffers are typically water-based.
Application of an electric current to highly conductive electrophoretic media and buffers produces large amounts of heat. If not dissipated or reduced, this heat can interfere with the separation process, destroy the molecules being separated, and damage the electrophoretic equipment. Heat generation can be reduced by applying a lower voltage across the electrodes of the electrophoresis unit. However, using a lower voltage increases the overall separation time for the molecules. Alternatively, the heat can be dissipated by using a large volume of electrophoresis buffer as a heat sink, or by direct cooling of the electrophoresis buffer. Either of these techniques increases the cost, complexity and size of the electrophoretic separation apparatus.
Aqueous electrophoresis media are also unsuited for separating hydrophobic proteins (e.g., biologically important cell membrane proteins) and some low molecular weight proteins (e.g., Mr≦10,000). Such proteins could be separated using organic solvent buffers. As organic solvent buffers are typically of low to medium conductivity, the problems of heat generation discussed above might also be alleviated. However, difficulties in polymerizing some gels in organic solvents, and the incompatibility of organic solvent buffers with many gel electrophoresis systems, have greatly limited the use of such buffers in protein electrophoresis.
A 1-D electro-separation has been developed which uses water-miscible organic solvents to separate small molecules on separation substrates such as filter paper (see U.S. Pat. No. 4,146,454; Haber N., PNAS USA, 79:272-276, 1982; and Haber N., Biotechnic & Histochemistry, 73: 59-70, 1998). This system is called “electro-molecular propulsion” or “EMP.” In EMP, nonpolar or uncharged compounds (such as aromatic hydrocarbons) are induced to migrate through the separation substrate once a threshold current level is passed.
Unlike conventional electrophoresis systems, movement of molecules by EMP does not depend on ionic species dissolved in an electrolytically conductive medium. See Haber N., Biotechnic & Histochemistry, 1998, supra. Rather, EMP induces the migration of nonpolar or uncharged compounds by “charge transfer” effects that impose electronic charges on the molecules by an unknown mechanism. The EMP “charge-induced” molecules respond electrokinetically to an applied electrical field, resulting in migration of the molecules.
The EMP technique appears useful for separating small nonpolar molecules such as dye compounds. However, it is not clear whether EMP is suitable for analysis of ampholytic biopolymers such as proteins, even though albumin, hemoglobin, myoglobin, cytochrome C and chymotrypsinogen have been separated on Whatman No. 3 filter paper using this technique (Haber N., PNAS USA, 1982, supra). Also, the filter papers and other substrates used in the EMP process do not bind proteins well, and proteins separated by EMP begin to diffuse on the substrates almost immediately after cessation of the electric current. The diffusion of proteins has greatly limited the usefulness of the EMP process, and no 2-D protein separation procedure employing filter papers has been reported.
Proteins separated by conventional electrophoretic techniques are often “blotted” or transferred onto high protein binding capacity, low porosity membranes made from nitrocellulose, nylon, polyvinylidene difluoride (PVDF) or other protein-binding polymers. The blot membranes are then subjected to staining, immunodetection (e.g., Western blot), mass spectrometry, amino acid sequence analysis and other operations. The blotting step is time consuming, and can result in an inefficient transfer of the separated proteins. For example, the retention of low molecular weight proteins by nitrocellulose is influenced by the presence of methanol in the transfer buffer (Pluskal et al., Biotechniques 4:272-283, 1986). Higher molecular weight proteins are also known to have lower transfer efficiency onto blotting membranes.
What is needed, therefore, is a high speed, high resolution electrophoresis system that employs organic solvent buffers compatible with hydrophilic, hydrophobic and low molecular weight proteins. The organic solvent buffers should preferably be non-denaturing to preserve protein binding interactions and biological activities, and should have low conductivity so as to minimize heat generation during electrophoretic separation. What is also needed is a separation substrate which minimizes diffusion of the molecules after electrophoresis is completed, and which eliminates the need for transferring the separated molecules from the separation matrix onto a blotting membrane.